Search

Cell communication is crucial for regulating cell growth, proliferation, survival, and function. The process of cell communication and cell signaling involves the recognition of exogenous or endogenous signaling molecules by receptors expressed on the surface of the plasma membrane. The activation of surface receptors such as enzyme-linked receptors results in the activation of the receptor's intracellular domain, which might have an intrinsic kinase activity like receptor tyrosine kinase (RTK). The activation of RTK triggers multiple downstream signaling pathways that regulate cell proliferation, angiogenesis, apoptosis, motility, adhesion, and metastasis. Therefore, a thorough understanding of RTK activation mechanisms and subsequent signaling events is crucial to provide insights into the control of RTK activity. The work presented in this dissertation is studying two different receptors from two subfamilies of RTKs, c-KIT, and EGFR/HER receptors family.The first project in this dissertation is focused on studying the SCF-mediated c-KIT signaling in mast cells (MCs) and the role of the underlying mechanisms in modulating mast cell viability and proliferation. The number of MCs in tissues usually remains constant, inflammation and asthma disturb this homeostasis, leading to proliferation of MCs. Understanding the signaling events behind this proliferative response could lead to the development of novel strategies for better management of allergic diseases. MC survival, proliferation, differentiation, and migration are all maintained by a MC growth factor, stem cell factor (SCF) via its receptor, KIT. Here, we explored how protein kinase C (PKC) redundancy influences MC proliferation in bone marrow-derived MC (BMMC). We found that SCF activates PKCα and PKCβ isoforms, which in turn modulates KIT phosphorylation and internalization. Further, PKCα and PKCβ activate p38 mitogen-activated protein kinase (MAPK), and this axis subsequently regulates SCF-induced MC cell proliferation. To ascertain the individual roles of PKCα and PKCβ, we knocked down either PKCα or PKCβ or both via short hairpin RNA (shRNA) and analyzed KIT phosphorylation, p38 MAPK phosphorylation, and MC viability and proliferation. To our surprise, the downregulation of neither PKCα nor PKCβ affected MC viability and proliferation. In contrast, blocking both PKCα and PKCβ significantly attenuated SCF-induced cell viability and proliferation, suggesting that PKCα and PKCβ compensate for each other downstream of SCF signaling to enhance MC viability and proliferation. Our results not only suggest that PKC classical isoforms are novel therapeutic targets for SCF/MC-mediated inflammatory and allergic diseases, but they also emphasize the importance of inhibiting both PKCα and β isoforms simultaneously to prevent MC proliferation.One important step in the process of RTK activation is the receptor dimerization and interaction following the ligand binding. For years, structural and biochemical studies have been conducted to study the dimerization across the different members of the RTK family. While the dimerization of some receptors is ligand-dependent, others exhibit ligand-independent dimerization where the ligand does not participate in the dimerization interface. One important example of such interactions includes the oligomerization of the EGFR/HER receptor family. The second project studies the association between EGFR and HER2 as one of the most prevalent interactions across the HER family. Their heterotypic interaction was quantified using a two-color, time-resolved pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) in live COS-7 cells. Additionally, we used PIE-FCCS to study the effect of the oncogenic HER2S310F mutation on the EGFR/HER2 heterodimerization. Our results showed a significant increase in EGFR/HER2WT association following EGF treatment. This could be demonstrated by a significant increase in the fraction cross-correlation (fc) and a decrease in the effective diffusion coefficients (Deff) of both EGFR and HER2. However, in the case of EGFRWT/HER2S310F, a significant association between EGFR and HER2S310F mutant was observed before EGF, which increased significantly following EGF treatment. This work illustrates how PIE-FCCS can be used as a robust platform for investigating molecular interactions in biologically relevant conditions. Moreover, our Study suggests that HER2S310F strongly promotes the formation of ligand-independent EGFR/HER2 heterodimers that develop into heteromultimeric clusters with EGF ligand treatment. This finding could be an intriguing target for designing improved therapies for cancers expressing both proteins, as well as cancers that harbor HER2S310F oncogenic mutation.

Lung cancer is the leading cause of cancer related mortality in the United States, accounting for more than 142,000 estimated deaths in 2019. The major subtype of lung cancer is non-small cell lung cancer (NSCLC), which represents 85% of all cases. Despite the advancement in understanding the molecular basis of NSCLC, the 5-year survival rate is less than 20%. The current treatment strategies for advanced stage patients rely on molecularly-targeted therapies, cytotoxic chemotherapy and immunotherapeutic agents. Because of the complexity and heterogeneity of lung tumors, intrinsic and acquired resistance mechanisms ultimately result in failure to respond to these therapies and early relapse. Combinatorial strategy that involves targeting multiple aspects of tumorigenesis may represent a new avenue for therapeutic intervention in lung cancer management. Estrogen signaling has been frequently shown to be an important mediator of lung cancer progression and metastasis. In a non-genomic fashion, ER mutually interacts with human epidermal growth factor receptors (HERs) to promote lung cancer proliferation and growth. Targeting ER signaling with the antiestrogen fulvestrant has shown moderate activities in preclinical models of lung cancer. The reciprocal interaction between ER and EGFR could limit the activity of using anti-ER agent alone. In a phase II clinical trial, combining fulvestrant with the selective EGFR tyrosine kinase inhibitor erlotinib showed an enhanced activity over erlotinib alone and improved the survival outcomes in NSCLC patients. However, the improvement in overall median survival was modest. Recent retrospective clinical analysis demonstrated that genes contained in the prediction analysis of microarray 50 (PAM50) provide prognostic information in high ERβ+ lung cancer patients. The 7-gene model includes c-Myc, MIA, CXXC5, FGFR4, Grb-7, FOXC1, and PgR. In high-risk patients, who tend to have a poor prognosis and short median survival, c-Myc, MIA, CXXC5, FGFR4, Grb-7, FOXC1 are overexpressed and PgR is downregulated. Importantly, the 7-gene model described one interacting network that includes ER and HER2/HER3 as the top upstream regulators for the 7-gene panel. In fact, a significant association between ERβ and HER2 expression was found, in which 70.2 % of ERβ-positive cases were positive for HER2 compared to 34.5% of ERβ-negative cases. HER3, when analyzed with HER2, showed also positive association with ERβ. These observations suggest that ERβ and HER2/HER3 pathways define lung tumors with very aggressive biology, indicating that blocking both pathways could be more efficacious than either one of them alone. These observations also could explain why the magnitude of response was modest when fulvestrant was combined with erlotinib, as erlotinib has weak activities against HER2 and HER3. Combining a pan-HER inhibitor such as dacomitinib (inhibits EGFR, HER2 and HER4) with fulvestrant could be more efficacious than either agent alone and could produce a gene signature that predicts better clinical outcomes in NSCLC. Immune checkpoint inhibitors have changed the treatment paradigm for several solid tumors, including lung cancer. Antibodies that target programmed death receptor 1(PD1) or its ligand (PD-L1) have proven great efficacy for certain patients with lung cancer. Patients who respond to these agents tend to have durable effects and longer survival outcomes; however, only 20-29% of patients are predicted to respond. Several combination strategies are being evaluated clinically to maximize the efficacy of these agents and improve the response rate. Preclinical evidence found that the ER blocker fulvestrant effectively sensitizes lung cancer cells to T cell and natural killer (NK) cell mediated cytotoxicity effects. In addition, estrogen signaling is a critical mediator for myeloid derived suppressor cells (MDSCs), which are largely known for their immunosuppressive effects. Evidence also demonstrated that EGFR inhibitors possess dual immunomodulatory effects that include upregulation of major histocompatibility complex I and II (MHC I and II), inducing the recruitment of immune cells, and inhibiting other tyrosine kinases essential for immune cells function. Synergy was observed when EGFR TKI and PD1 inhibitor was combined in an- EGFR-mutant model. Altogether, these previous observations encouragingly support the hypothesis that use of triple therapy (fulvestrant, pan-HER TKI and an immune checkpoint blocker) will be a promising treatment approach for lung cancer. Testing this hypothesis is the subject of this dissertation. To evaluate the therapeutic potential of combining ER blocker with pan-HER in NSCLC, we chose fulvestrant, as an antiestrogen, and dacomitinib, as a tyrosine kinase inhibitor that targets EGFR, HER2, and HER4. We first evaluated the efficacy of this combination in three different human NSCLC cell lines and assessed the ability of this treatment approach to produce a gene signature that predicts better clinical outcomes. We utilized three different cell lines that represent three different categories of NSCLC population; EGFR-mutant, KRAS-mutant and EGFR and KRAS wild-type. Next, we investigated the immunomodulatory effects of this combination treatment on macrophages and CD8+ T cells in vitro. We used a novel syngeneic lung cancer model (FVBW-17/FVB-N) to evaluate the therapeutic effectiveness of fulvestrant-dacomitinib in combination with a mouse anti-PD1. Major results The combination of fulvestrant and dacomitinib significantly suppressed NSCLC cell growth in vitro and produced a combination index < 0.5, indicating strong synergy. The combination also showed potent downregulation of HER activity and marked decrease in amphiregulin (AREG) and neuregulin (NRG1-β1) expression. Importantly, the combination, but not single agents, completely reversed the gene signature associated with poor prognosis. C-Myc, MIA, CXXC5, FGFR4, FOXC1 and Grb7 were downregulated and PgR was upregulated following the combination treatment. The combination significantly reduced c-Fos, JunB and pCREB DNA-binding activities. The c-Fos/Ap-1 inhibitor t-5224, but not CBP-CREB inhibitor, was able to partially mimic the effects of the combination in reversing the gene signature. In vivo, the combination treatment demonstrated a robust synergistic antitumor effect in NSCLC cell lines that were engrafted subcutaneously in immunodeficient mice. Tumor regression was observed in the majority of tumors following the combination treatment. A drastic decrease in HER activity and downstream signaling were observed with the combination, along with a significant decrease in AREG and NRG1-β1 expression. In situ proximity ligation assay revealed a significant decrease in the active dimerization of both p-HER2/p-HER3 and p-HER2/p-EGFR dimers following the combination treatment. Additionally, the gene signature was also completely reversed by the combination but not with single agents. In the EGFR mutant model, the survival of mice was improved after treatment discontinuation, tumors that recurred were less aggressive, and two mechanisms of resistance commonly associated with HER TKIs were blocked. To evaluate the immunomodulatory effects of the combination, bone marrow-derived macrophages and CD8+ T cells were treated with the combination. Surprisingly, macrophages lost their phagocytic function and behave more like M2-macrophages by expressing high IL-10, CD206 and PD1 following the combination treatment. Mechanistically, dacomitinib induced downregulation of phospho-Syk, and fulvestrant was not able to overcome this effect. In CD8+ T cells, the combination impaired the function of T cells, induced high PD1 expression, and severally reduced IFN-ϒ and TNF-α production. These debilitating effects were mostly attributed to the downregulation of Src Family kinases activities, as less phospho-SFK Y416 was detected following the combination treatment. In vivo, adding anti-PD1 antibody to the combination treatment enhanced the immune function and improved the antitumor effects. In a sequential approach, where anti-PD1 was administrated after the combination treatment, the average tumor volume was 4-fold less than placebo and this effect was synergistic. In comparison, the triple therapy given concomitantly showed a 2-fold decrease compared to placebo. Sequential triple therapy was also significantly better than concomitant triple therapy. None of the drugs alone show any sign of activities. Interestingly, after one week of administrating fulvestrant and dacomitinib, tumors showed high immune cell infiltration (inflamed tumor microenvironment), with relatively high PD1 expression on CD8+ T cells. In contrast, the concomitant triple therapy showed a significant increase in CD8+ T cells infiltration but with a decrease in PD1 expression, and fewer M2 macrophages. Conclusion and Significance Retrospective analysis of NSCLC patients revealed that the interaction between HER2/HER3 and ERβ contributes to poor outcomes in NSCLC patients with high ERβ expression. Here, we report that targeting ERβ with the antiestrogen fulvestrant, along with targeting multiple HER pathways with the pan-HER TKI dacomitinib produced synergistic antitumor effects in preclinical models of human ERβ+ NSCLC. The robust antitumor effect seen was accompanied by the ability of this combination treatment to produce a gene signature that predicts better clinical outcomes. The 7-gene model could serve as a predictive tool for identifying patients who will more likely respond to the treatment. These data strongly support its clinical use, giving the fact that both drugs are clinically used for cancer patients. Convincing evidence also suggest an immunomodulatory effect of ER signaling and HER pathways. The combination of fulvestrant and dacomitinib suppressed phagocytic macrophages and CD8+ T cell functions and upregulated PD1 expression. These effects were largely contributed by dacomitinib for its inhibitory effects on Syk and Src kinases on immune cells and might limit the clinical utility of the fulvestrant/dacomitinib combination. However, the ability of the combination to turn the TME into an inflamed microenvironment with upregulated PD1 could potentiate the tumor-mediated killing effects by immunotherapy. The synergistic antitumor effect observed with adding anti-PD1 in a sequential manner after the administration of fulvestrant and dacomitinib is highly encouraging. The significance of these observations is that the triple therapy has shown excellent antitumor effects in a highly aggressive tumor model that is unresponsive to immune checkpoint inhibitor alone or fulvestrant-dacomitinib combination. The clinical usefulness of this approach can have therapeutic applications for other solid tumor models, particularly in tumors that are less inflamed and are unlikely to respond to immune checkpoint inhibitors.

There are thirteen known endogenous EGF-like ligands. We previously reported that Betacellulin (BTC) increases ligand-mediated corneal wound healing more than Epidermal Growth Factor (EGF) [Peterson et al. (2014) IOVS 55(5):2870-80], although the molecular reason for this is unknown. Despite being better at promoting wound healing via enhanced cell migration, BTC has reduced receptor affinity and weaker induction of EGFR phosphorylation. These data indicate that BTC’s response is not due to enhanced affinity or EGFR-kinase activity. Receptor phosphorylation and proximity ligation assays indicate that BTC treatment significantly increases ErbB3 phosphorylation and EGFR:ErbB3 heterodimers. BTC traffics EGFR at a faster rate than EGF, without noticeable differences in effector signaling. Thus, we demonstrate that despite both ligands binding to the EGFR, BTC biases the EGFR to dimerize with ErbB3 and regulates the biological response through trafficking and unknown effectors.

Cancer is the most fatal disease after cardiovascular disease with over 8.2 million deaths worldwide each year. Ever since the serendipitous discovery of mustard gas as an anti-cancer therapeutic in the 1940s, serious efforts have been put into discovering more chemotherapies. Chemotherapies can be categorized into different groups such as alkylating agents (cisplatin, cyclophosphamide), antimetabolites (5-fluorouracil, Ara-C) and mitotic inhibitors (taxanes, vinca alkoids) among others. While chemotherapies have proven to kill cancer cells by targeting cell division processes, over time, tumor cells can adapt and become resistant to these drugs. With a growing understanding of cell signaling networks, targeted therapies are being developed to overcome the issue of chemotherapy resistance. Targeted therapies are highly specific molecules that bind to a specific cellular protein or molecule and block signaling networks associated with biological processes. One of the most frequently dysregulated receptor systems in cancers is the receptor tyrosine kinase family with ErbB being one of the most studied receptors families. ErbB or HER receptors consists of four structurally related receptor tyrosine kinases namely, EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4. The ErbB family of receptors plays a major role in morphogenesis of the human body as well as various cellular responses such as cell growth, differentiation and proliferation. Overexpression and dysregulation of these receptors, particularly EGFR and HER2, have been linked to a number of cancers such as breast cancer, gastric cancer, ovarian cancer and non-small cell lung cancer, to name a few. One of the most successful therapies against ErbB related cancers have been targeted therapies. Targeted therapies for ErbB related cancers are of two kinds: (i) Small molecule tyrosine kinase inhibitors (such as erlotinib and gefitinib against EGFR) and, (ii) Monoclonal antibodies (such as trastuzumab against HER2 and cetuximab against EGFR). These drugs function either by inhibiting the kinase activity of the receptor and preventing phosophorylation of tyrosine residues, or binding to some other site on the extracellular domain of the receptor and preventing ligand binding and heterodimerization of ErbB monomers. These drugs have proven to have limited efficacy as monotherapy, but are more effective in combination with standard chemotherapies. However, tumor cells can adapt their signaling networks developing resistance to targeted therapies over the course of treatment and lead to cancer progression. While overexpression and dysfunction of EGFR and HER2 are implicated in most ErbB driven cancers, recent studies have found HER3 playing a pivotal role in inducing resistance to EGFR and HER2 targeted therapies in various cancers and has been found to be the most sensitive node in driving the PI3K pathway leading to tumorigenesis. Thus, there is an urgent need to develop drugs targeted against HER3 and bring them into the clinic. Since HER3 lacks kinase activity, only monoclonal antibodies can be developed against it. Currently, there are a number of molecules in clinical development that target HER3. For example, patritumab and MM-121 are humanized monoclonal antibodies that target the extracellular domain of HER3 receptor and leads to inhibition of HER3-PI3K signaling followed by rapid internalization of the receptor. MM-111 and MM-141, two different bispecific monoclonal antibodies that bind to HER2, HER3 and IGFR-1, HER3, respectively, are currently in clinical development. HER3 inhibitors provide hope to effectively overcome HER3 induced tumor resistance and successfully treat several ErbB driven cancers. However, further development of HER3 inhibitors is necessary by taking strategic approaches. One of these approaches it the utilization of systems biology, a branch of biology that involves computational and mathematical modeling of complex biological systems with the aim of discovering emergent properties of biological systems. Systems biology enables researchers to get a deeper understanding of biological networks such as that of ErbB and make predictive models and test outcomes. This approach was used by Merrimack Pharmaceuticals to develop novel monoclonal antibodies against HER3. Computational outcomes were successfully validated by in vitro and in vivo experiments. Thus, this suggests that systems biology might be the future of designing and developing HER3 inhibitors that would successfully overcome HER3 resistance and cancer progression.

The erbB1 receptor regulates cellular programs including proliferation, migration, and differentiation and is the prototypical receptor tyrosine kinase (RTK). The erbB family consists of four homologous transmembrane receptors (erbB1/HER1/EGFR, erbB2/HER2, erbB3/HER3, erbB4). Canonically, ligand binding leads to an extracellular conformational change that promotes the formation of a receptor-mediated back-to-back dimer, asymmetric orientation of the catalytic kinase domains, and downstream transphosphorylation of cytoplasmic tyrosine residues. Exceptions to this paradigm are the orphan erbB2 and the kinase defective erbB3. The erbB receptors are implicated in mechanisms of carcinogenesis and are, thus, important therapeutic targets. Antibodies and small molecule inhibitors have been used to target cancer cells expressing erbB1 and erbB2, however, tumors often become resistant to treatment. Recent evidence implicates erbB3 in escape from erbB1- and erbB2-targeted pharmacological agents. Therefore, understanding the function of these receptors and their interactions with each other is important for designing better therapeutics. Here, we investigated erbB dimer formation and lifetime using live cell imaging and an analytical three-state Hidden Markov Model (HMM). First, multi-color quantum dot (QD) based probes that label resting or activated receptors were used to directly observe dimerization and quantify diffusion and correlated motion. Second, pairwise analyses of single particle trajectories in our HMM are used to characterize transition rates between free, confined, and dimerized states. We examined preformed, unliganded erbB1 homodimers and demonstrate that these do not display correlated motion and that observed dimers are short lived. Interestingly, liganded erbB1 dimers have the same off rate regardless of the activation status of the kinase domain. We further describe features of membrane organization, in particular demonstrating differential partitioning of activated receptors that alters mobility and permits repeated interactions within domains. Important mechanistic insight comes from our novel observations of short lived erbB2-erbB3 heterodimers and long lived erbB3 homodimers. Prior biochemical studies suggested that the erbB2-erbB3 heterodimer was the functional signaling unit. Our single particle tracking results are consistent with a new model for an active erbB3 kinase domain that is dependent on interactions with erbB2. Furthermore, our data indicate that erbB3 dimers and, ultimately, oligomers may be the principal signaling complex. This work demonstrates the importance of membrane architecture and reorganization in signal transduction and sheds new light on mechanisms of erbB activation with unprecedented spatial and temporal resolution.